CN101974637A - Kit for detecting susceptibility of lung cancer and judging prognosis of lung cancer by MAPKAPK2 gene and application thereof - Google Patents
Kit for detecting susceptibility of lung cancer and judging prognosis of lung cancer by MAPKAPK2 gene and application thereof Download PDFInfo
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- CN101974637A CN101974637A CN2010105388759A CN201010538875A CN101974637A CN 101974637 A CN101974637 A CN 101974637A CN 2010105388759 A CN2010105388759 A CN 2010105388759A CN 201010538875 A CN201010538875 A CN 201010538875A CN 101974637 A CN101974637 A CN 101974637A
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Abstract
The invention discloses a kit for detecting susceptibility of lung cancer and judging prognosis of lung cancer by MAPKAPK2 gene and an application thereof, which is characterized in that 1) the materials of the kit comprise a specific primer pair and a specific fluorescent probe pair for detecting SNP locus of No. rs4844550 on the MAPKAPK2 gene, Taq DNA polymerase, dNTP mixed liquid, MgCL2 solution, fluorescence quantitative PCR reaction buffer solution and deionized water; 2) the sequences of the specific primer pair for detecting the SNP locus of No. rs4844550 on the MAPKAPK2 gene comprise a sense primer sequence and an antisense primer sequence; the specific fluorescent probe pair for detecting the SNP locus of No. rs4844550 on the MAPKAPK2 gene comprises a wild type probe sequence and a mutant type probe sequence; and 3) the specific primer can amplify a DNA primer pair and a DNA probe pair aiming at the SNP locus of No. rs4844550 on the MAPKAPK2 gene specifically. In the invention, the susceptibility of individual lung cancer is detected by detecting the SNP locus of No. rs4844550 on the MAPKAPK2 gene, and the prognosis of a patient with lung cancer can be evaluated; and the kit of the invention can be widely applied in medical research.
Description
Technical field
The present invention relates to the test kit that a kind of disease susceptibility detects usefulness, especially a kind of by MAPKAPK2 gene test lung cancer susceptibility and test kit of judging lung cancer for prognosis and uses thereof, by detecting MAPK activated protein kinase-2 (MAPK activated protein-kinase 2, MAPKAPK2) susceptibility of lung cancer is predicted in single nucleotide polymorphism (SNP) site, and can judge the prognosis of patients with lung cancer.Belong to the medical research field.
Background technology
Primary bronchogenic carcinoma of lung is called for short lung cancer, is one of malignant tumour that M ﹠ M is the highest in the world at present.Because early stage transfer of the normal companion of lung cancer, 70% lung cancer patient has been late period when finding, its 5 years present mean level (ML)s of survival rate have only 16%, and its PD speed is relevant with the biological nature of lung carcinoma cell.Lung cancer is the disease of a kind of serious threat human health and life.U.S.'s report New Development lung cancer 219,440 examples in 2009, dead 159,390 examples (mortality ratio 53.80/10 ten thousand).The data of Guangzhou record of deaths are indicated: lung cancer mortality was 11.61/10 ten thousand in 1970～1972 years, and 1980～nineteen eighty-two is upgraded to 32.67/10 ten thousand, and more rises to 48.79/10 ten thousand in 2000～2002 years.The data of big city such as Shanghai, Tianjin record of deaths show all that also the mortality ratio of lung cancer sharply rises between many decades.
Pulmonary carcinosis because of and pathogenesis fully clear and definite as yet.At present consistently think that occupational hazards such as smoking, atmospheric pollution, radioactive rays, pulmonary tuberculosis and nickel compound are risk factors of lung cancer.These Hazard Factor pair cells all are stimulus informations, can activate mitogen activated protein kinase (mitogen-activated protein kinase, MAPK) signal transduction pathway.Processes such as the further regulating cell apoptosis of MAPK signal path, inflammatory reaction and cell proliferation cause tumour to take place.Recent study shows that a lot of MAPK genes all have the change of genetic expression in all kinds lung cancer, and has found the sudden change of a lot of MAPK genes in nonsmall-cell lung cancer.Further investigation to the MAPK gene will be provided fundamental basis for the gene prevention and the treatment of lung cancer.
SNP (Single nucleotide polymorphism), being single nucleotide polymorphism, is the third generation genetic marker of human genome mapping, mainly is meant the dna sequence polymorphism that a class causes based on single nucleotide variation, comprise single base conversion, transversion, and the insertion/disappearance of single base etc.It is class polymorphism mark of extensive existence of genome, has become a kind of instrument of research genome polymorphism and identification, location disease-related.Successfully developed the real-time fluorescence quantitative PCR technology that SNP detects in 1996, realized the leap of PCR, also realized the automatism of judged result from qualitative to quantitative.Can utilize the fluorescent quantitative PCR technique research and development to detect the test kit of disease susceptibility and judgement disease prognosis.
The MAPKAPK2 gene is positioned at karyomit(e) 1q32 position No. 1, always is about 49kb, is made up of 10 exons, 400 the amino acid whose MAPKAPK2 protein of encoding.The MAPKAPK2 gene coded protein is a multi-functional protein kinase, is the important effect substrate of MAPK.It can pass through to regulate cancer suppressor protein tuberin, and functions such as cell cycle regulation and iuntercellular adhesion; Can influence the anti-oxidant function of pulmonary epithelial cells; Also but heat of activation shock protein HSP27, HSP25 suppress apoptosis and cell movement; Also can influence scavenger cell and dendritic cell function, regulate the resistibility of body the external source infection by the secretion of regulating TNF etc.Under the situation of irradiation damage, MAPKAPK2 can regulate the cell cycle check point of DNA-repair gene ATM and/or ATR startup, influences the reparation of body cell to dna damage.In addition, in the animal lung cancer model, find the too high key character that is expressed as the change of lung tissue pathology with MAPKAPK2.Prompting MAPKAPK2 can participate in the process of cell carcinogenesis by a plurality of approach.Moreover, MAPKAPK2 still is a kind of important cells cycle detection point regulation and control of discovered in recent years, influences the effect of cis-platinum, camptothecine and Zorubicin killing tumor cell; It also can be by activating the LIM molecule, and participating in VEGF stimulates the tumor-blood-vessel growth mistake that causes.Prompting MAPKAPK2 may be relevant with prognosis of lung cancer.
Rs4844550 is that to be positioned at the A＞G of MAPKAPK2 gene promoter region of No. 1 karyomit(e) 1q32 polymorphic.It is in world crowd's frequency distribution, and A accounts for 0.91, and G accounts for about 0.09.The distribution A of Chinese population accounts for 0.93, and G accounts for about 0.07.Molecular biology research shows that the alternative MAPKAPK2 of the causing gene promoter of A＞G has lost transcription factor-aromatic hydrocarbon receptor supressor, and (AHR repressor, AHRR) bonded site has increased the MAPKAPK2 expression of gene.By the lung cancer case control study of large-scale crowd, the A＞G of MAPKAPK2 gene promoter region is polymorphic to be taken place relevantly with lung cancer, thisly is associated in youth＜60 years old person and has tumour family history person particularly evident.The heritable variation of finding this site in addition is relevant with the prognosis of patients with lung cancer, when the patients with lung cancer genotype carries G, and poor prognosis.The polymorphic susceptibility that can be used for detecting individual lung cancer of this SNP, and can be used for judging the prognosis of patients with lung cancer.
Summary of the invention
First purpose of purpose of the present invention, being for a kind of test kit by MAPKAPK2 gene test lung cancer susceptibility and judgement lung cancer for prognosis is provided, is a kind of by rs4844550 site detection of lung cancer susceptibility on the MAPKAPK2 gene and the test kit of judging lung cancer for prognosis.
Second purpose of the present invention is in order to provide a kind of by the purposes of MAPKAPK2 gene test lung cancer susceptibility with the test kit of judging lung cancer for prognosis.
First purpose of the present invention can be achieved through the following technical solutions:
Test kit by MAPKAPK2 gene test lung cancer susceptibility and judgement lung cancer for prognosis is characterized in that:
1) material of this test kit comprises: the Auele Specific Primer that detects the rs4844550 SNP site on the MAPKAPK2 gene to and the specificity fluorescent probe to, Taq archaeal dna polymerase, dNTP mixed solution, MgCl2 solution, quantitative fluorescent PCR reaction buffer and deionized water;
2) the right sequence of Auele Specific Primer in the rs4844550 SNP site on the described detection MAPKAPK2 gene includes adopted primer sequence and antisense primer sequence; The specificity fluorescent probe in the rs4844550 SNP site on the described detection MAPKAPK2 gene is to comprising wild-type probe sequence and mutant probe sequence;
3) described Auele Specific Primer can specific amplification goes out to comprise dna primer at the rs4844550 SNP site on the MAPKAPK2 gene to right with dna probe.
A kind of embodiment that realizes the present invention's first purpose is: the content of each composition that uses for a person-portion in the test kit is:
1) 10 μ M Auele Specific Primers are to there being each 0.225 μ l of adopted primer sequence and antisense primer sequence, and 5 μ M specificity fluorescent probes are to wild-type probe sequence and each 0.1 μ l of mutant probe sequence;
2) 5unit/ μ lTaq archaeal dna polymerase 0.02 μ l;
3) 20mM dNTP mixed solution 0.1 μ l;
4) 25mM MgCl2 solution 0.5 μ l;
5) 5X quantitative fluorescent PCR reaction buffer 2 μ l;
6) deionized water 4.1 μ l.
A kind of embodiment that realizes the present invention's first purpose is: the right sequence of the Auele Specific Primer in the rs4844550 SNP site on the described detection MAPKAPK2 gene is as sequence table SEQ ID №: shown in 1, the Tm value that adopted primer sequence is arranged is 59 ℃, and the Tm value of antisense primer sequence is 58 ℃; The right sequence of the specificity fluorescent probe in the rs4844550 SNP site on the described detection MAPKAPK2 gene is as sequence table SEQ ID №: shown in 2, the Tm value of wild-type probe sequence is 68 ℃, and the Tm value of mutant probe sequence is 68 ℃.
A kind of embodiment that realizes the present invention's first purpose is: the storage temperature of test kit is-20 ℃.
First purpose of the present invention can be achieved through the following technical solutions:
By the test kit of MAPKAPK2 gene test lung cancer susceptibility and judgement lung cancer for prognosis, its thing is levied and is: the genotype that is used to detect the rs4844550 SNP site on the MAPKAPK2 gene.
The present invention has following outstanding beneficial effect:
1, The present invention be directed to the rs4844550 SNP site on the MAPKAPK2 gene and design, can be by regulating cancer suppressor protein tuberin, and functions such as cell cycle regulation and iuntercellular adhesion; Can influence the anti-oxidant function of pulmonary epithelial cells; Also but heat of activation shock protein HSP27, HSP25 suppress apoptosis and cell movement; Also can influence scavenger cell and dendritic cell function, regulate the resistibility of body the external source infection by the secretion of regulating TNF etc.
2, the heritable variation in the rs4844550 SNP site on the MAPKAPK2 gene of the present invention is relevant with the prognosis of patients with lung cancer, the polymorphic susceptibility that can be used for detecting individual lung cancer of this SNP, and can be used for judging the prognosis of patients with lung cancer, can be widely used in medical research.
Embodiment
Specific embodiment 1:
Present embodiment comprises:
1) material of this test kit comprises: the Auele Specific Primer that detects the rs4844550 SNP site on the MAPKAPK2 gene to and the specificity fluorescent probe to, Taq archaeal dna polymerase, dNTP mixed solution, MgCl2 solution, quantitative fluorescent PCR reaction buffer and deionized water;
2) the right sequence of Auele Specific Primer in the rs4844550 SNP site on the described detection MAPKAPK2 gene includes adopted primer sequence and antisense primer sequence; The specificity fluorescent probe in the rs4844550 SNP site on the described detection MAPKAPK2 gene is to comprising wild-type probe sequence and mutant probe sequence;
3) described Auele Specific Primer can specific amplification goes out to comprise dna primer at the rs4844550 SNP site on the MAPKAPK2 gene to right with dna probe.
In the present embodiment, the right sequence of the Auele Specific Primer in the rs4844550 SNP site on the described detection MAPKAPK2 gene is as sequence table SEQ ID №: shown in 1, the Tm value that adopted primer sequence is arranged is 59 ℃, and the Tm value of antisense primer sequence is 58 ℃; The right sequence of the specificity fluorescent probe in the rs4844550 SNP site on the described detection MAPKAPK2 gene is as sequence table SEQ ID №: shown in 2, the Tm value of wild-type probe sequence is 68 ℃, and the Tm value of mutant probe sequence is 68 ℃.
The content of each composition that uses for a person-portion in the test kit is:
1) 10 μ M Auele Specific Primers are to there being each 0.225 μ l of adopted primer sequence and antisense primer sequence, and 5 μ M specificity fluorescent probes are to wild-type probe sequence and each 0.1 μ l of mutant probe sequence; 2) 5unit/ μ lTaq archaeal dna polymerase 0.02 μ l; 3) 20mM dNTP mixed solution 0.1 μ l; 4) 25mM MgCl2 solution 0.5 μ l; 5) 5X quantitative fluorescent PCR reaction buffer 2 μ l; 6) deionized water 4.1 μ l.
The storage temperature of test kit is-20 ℃.
Application Example 1 of the present invention: the use of detection kit
The extraction of step 1:DNA template
Extract the genomic dna of blood preparation with silica gel adsorption.
Step 2: quantitative fluorescent PCR reaction
Use fluorescent quantificationally PCR detecting kit of the present invention, comprising following primer to and fluorescent probe, all at the rs4844550 SNP site on the MAPKAPK2 gene:
It is 59 ℃ that adopted primer 1:5 '-CTTCTATGCAGGGTCCAGCTAGA-3 ' (SEQ ID NO:1) Tm value is arranged
Antisense primer 1:5 '-GGAATGGCTTCTGATTTCCG-3 ' (SEQ ID NO:2) Tm value is 58 ℃
Wild-type probe: 5 '-GGGTTAGCCCCGTGTATC TC-3 ' (SEQ ID NO:3) Tm value is 68 ℃
The mutant probe: 5 '-GGCTAGCCCCGTGTATC TCA-3 ' (SEQ ID NO:4) Tm value is 68 ℃
Quantitative fluorescent PCR reaction system cumulative volume is 10 μ l, comprise dna profiling (20ng/ μ l) 2 μ l, 10 μ M Auele Specific Primers to (two) each 0.225 μ l, 5 μ M Auele Specific Primers to (two) each 0.1 μ l, (5unit/ μ l) Taq archaeal dna polymerase 0.02 μ l, 20mM dNTP mixed solution 0.1 μ l, 25mM MgCl2 solution 0.5 μ l, 5X quantitative fluorescent PCR reaction buffer 2 μ l, deionized water 4.1 μ l.
At ABI 7900HT type quantitative real time PCR Instrument is to react, and reaction conditions is 50 ℃ of 2 min, and 95 ℃ of 10min carry out 40～55 PCR circulations: 92 ℃ of 15s, 60 ℃ of 1min again.Reaction finishes the back and reads fluorescence volume at ABI 7900HT type quantitative real time PCR Instrument.
Step 3: gene type assay
Adopt ABI 7900HT type quantitative real time PCR Instrument to carry software SDS (Sequence Detection Systems) V2.3 interpretation genotype, automatically analytical results.When the rs4844550 SNP site on the MAPKAPK2 gene of detected DNA carries T, be lung cancer susceptible type.
Application Example 2 of the present invention: instruct people initiatively to prevent lung cancer
The extraction of step 1:DNA
The detected person is served preceding consulting, sign Informed Consent Form, fill in living and diet custom questionnaire table by the detected person.Ordinary method extracts detected person EDTA anticoagulation 5ml.Extract the genomic dna of blood preparation with silica gel adsorption.
Step 2: genotype tests
Use test kit provided by the invention, fluorescence quantitative PCR detection is carried out in the rs4844550 SNP site on the MAPKAPK2 gene of detected person DNA, determine the genotype in this site.
Step 3: instruct people initiatively to prevent lung cancer
By to the genotypic analysis of detected person, provide genotype tests and report and detected person's individuation health guidance is reported.The genotype tests report describes the height and the P size of detected person's lung cancer susceptible degree in detail.The report of individuation health guidance in conjunction with its living and diet custom survey result, is assessed the relative risk of detected this lung cancer inheritance susceptible based on detected person's genotype tests result.Make personalized healthy action scheme simultaneously, be included in the recommendation on improvement of food habits, mode of life life etc., and popularize the health knowledge of prevention lung cancer for the detected person at the detected person.
Application Example 3 of the present invention: instruct the clinical judgment lung cancer for prognosis
The extraction of step 1:DNA
Detected patients with lung cancer is served preceding consulting, sign Informed Consent Form, provide the patient basic clinical information by the clinician by the detected person.Ordinary method extracts detected person EDTA anticoagulation 5ml before chemotherapy.Extract the genomic dna of blood preparation with silica gel adsorption.
Step 2: genotype tests
Use test kit provided by the invention, fluorescence quantitative PCR detection is carried out in the rs4844550 SNP site on the MAPKAPK2 gene of detected person DNA, determine the genotype in this site.
Step 3: instruct people initiatively to prevent lung cancer
By to the genotypic analysis of detected person, provide the genotype tests report and detected person's individualized treatment is instructed report.The genotype tests report describes the quality and the survival probability size of detected patients with lung cancer prognosis in detail.Individualized treatment instructs the genotype tests result of report based on the detected person, in conjunction with its pathology somatotype, wait clinical information by stages, assesses detected detection of lung cancer patient's 3 years or 5 probabilities of living in a year.Make simultaneously at detected person's individualized treatment scheme, comprise whether performing the operation, chemicotherapy scheme and dosage etc.
Claims (5)
1. by the test kit of MAPKAPK2 gene test lung cancer susceptibility and judgement lung cancer for prognosis, it is characterized in that:
1) material of this test kit comprises: the Auele Specific Primer that detects the rs4844550 SNP site on the MAPKAPK2 gene to and the specificity fluorescent probe to, Taq archaeal dna polymerase, dNTP mixed solution, MgCl2 solution, quantitative fluorescent PCR reaction buffer and deionized water:
2) the right sequence of Auele Specific Primer in the rs4844550 SNP site on the described detection MAPKAPK2 gene includes adopted primer sequence and antisense primer sequence; The specificity fluorescent probe in the rs4844550 SNP site on the described detection MAPKAPK2 gene is to comprising wild-type probe sequence and mutant probe sequence;
3) described Auele Specific Primer can specific amplification goes out to comprise dna primer at the rs4844550 SNP site on the MAPKAPK2 gene to right with dna probe.
2. according to claim 1 by MAPKAPK2 gene test lung cancer susceptibility with judge the test kit of lung cancer for prognosis, it is characterized in that in the test kit for the content of each composition of person-portion use being:
1) 10 μ M Auele Specific Primers are to there being each 0.225 μ l of adopted primer sequence and antisense primer sequence, and 5 μ M specificity fluorescent probes are to wild-type probe sequence and each 0.1 μ l of mutant probe sequence;
2) 5unit/ μ lTaq archaeal dna polymerase 0.02 μ l;
3) 20mM dNTP mixed solution 0.1 μ l;
4) 25 mM MgCl2 solution, 0.5 μ l;
5) 5X quantitative fluorescent PCR reaction buffer 2 μ l;
6) deionized water 4.1 μ l.
3. the test kit by MAPKAPK2 gene test lung cancer susceptibility and judgement lung cancer for prognosis according to claim 1, it is characterized in that: the right sequence of the Auele Specific Primer in the rs4844550 SNP site on the described detection MAPKAPK2 gene is as sequence table SEQ ID №: shown in 1, the Tm value that adopted primer sequence is arranged is 59 ℃, and the Tm value of antisense primer sequence is 58 ℃; The right sequence of the specificity fluorescent probe in the rs4844550 SNP site on the described detection MAPKAPK2 gene is as sequence table SEQ ID №: shown in 2, the Tm value of wild-type probe sequence is 68 ℃, and the Tm value of mutant probe sequence is 68 ℃.
4. the test kit by MAPKAPK2 gene test lung cancer susceptibility and judgement lung cancer for prognosis according to claim 1, it is characterized in that: the storage temperature of test kit is-20 ℃.
5. by the purposes of MAPKAPK2 gene test lung cancer susceptibility with the test kit of judging lung cancer for prognosis, its thing is levied and is: the genotype that is used to detect the rs4844550 SNP site on the MAPKAPK2 gene.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267611A (en) * | 2017-06-26 | 2017-10-20 | 广州医科大学 | A kind of primer sets of detection p.Pro189Ala loci gene types and its detection kit and application |
| CN114410772A (en) * | 2021-01-28 | 2022-04-29 | 中国医学科学院北京协和医院 | Susceptibility gene of chronic obstructive pulmonary acute exacerbation and application thereof in predicting susceptibility to chronic obstructive pulmonary acute exacerbation |
-
2010
- 2010-11-09 CN CN2010105388759A patent/CN101974637A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267611A (en) * | 2017-06-26 | 2017-10-20 | 广州医科大学 | A kind of primer sets of detection p.Pro189Ala loci gene types and its detection kit and application |
| CN107267611B (en) * | 2017-06-26 | 2020-12-01 | 广州医科大学 | Primer group for detecting p.Pro189Ala locus genotype and detection kit and application thereof |
| CN114410772A (en) * | 2021-01-28 | 2022-04-29 | 中国医学科学院北京协和医院 | Susceptibility gene of chronic obstructive pulmonary acute exacerbation and application thereof in predicting susceptibility to chronic obstructive pulmonary acute exacerbation |
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Application publication date: 20110216 |